Leishmania (Leishmania) infantum DNA detection in Nyssomyia neivai in Vale do Ribeira, Paraná, Brazil

BACKGROUND The incidence of visceral leishmaniasis (VL) has increased in the Southern region of Brazil in recent years, especially in the State of Paraná. New species have been suggested with potential to act as vector in VL endemic areas. OBJECTIVES Identify the Leishmania species in sand fly specimens collected from 2016 to 2018 in the municipality of Itaperuçu, Vale do Ribeira, Paraná, Brazil. METHODS Light traps were used for collections and for the analysis of sand fly were used the multiplex polymerase chain reaction (PCR) methodology and subsequent sequencing. FINDINGS Among the collected specimens, 88.62% were attributed to the species Nyssomyia neivai, which were grouped into 176 pools. Three positive pools were detected: two with Leishmania (Viannia) braziliensis and one with L. (Leishmania) infantum. The positivity rate for the parasite was 0.25% based on the presence of at least one infected insect in the pool. MAIN CONCLUSIONS The detection of L. infantum in Ny. neivai draws attention due to its abundance and anthropophily in the State of Paraná. Moreover, this finding is considered as an alert and suggests that the vector competence of Ny. neivai and the criteria for its incrimination should be carried out, given its wide distribution in southern of Brazil.

Visceral leishmaniasis (VL), also called kala-azar, has been widely distributed in the Americas.¹ ) The etiological agent of VL in the Americas is the species Leishmania (Leishmania) infantum and its transmission is attributed to the species Lutzomyia (Lutzomyia) longipalpis and Lutzomyia (Lutzomyia) cruzi.However, in the last two decades, studies have presented evidence suggesting that other species of sand flies can function as vectors of L. infantum, such as Migonemyia migonei, (2) Pintomyia fischeri (3) and Nyssomyia neivai. (4,5)yssomyia neivai is the sand fly species that has the widest geographic distribution in southern Brazil (6) and is considered the main vector of American Tegumentary Leishmaniasis (ATL) in the State of Paraná. (6,7,8,9)According to Silva et al., (6) the species was found in high densities in forest, domicile, and peridomicile areas in Vale do Ribeira, which is an endemic region for ATL located in the states of Paraná and São Paulo, evidencing its wide distribution in the region.Ny. neivai has a high doi: 10.1590/0074-02760230173 LCM and DPP contributed equally to this work.+ Corresponding author: magdaribeiro@ufpr.br https://orcid.org/0000-0003-0215-2596Received 27 September 2023 Accepted 13 December 2023 capacity for adapting to modified environments where there is anthropic action and has already been found naturally infected by L. infantum. (4)In recent years, the incidence of VL has increased in the Southern region of Brazil, especially in the State of Paraná, where 25 cases of VL have been diagnosed in the last six years. (10)The municipality of Foz de Iguaçu has in its sand fly fauna the predominant species Lu. longipalpis, and it is where the third detection of the presence of DNA from L. infantum in Ny. neivai was reported. (11)This scenario increasingly reinforces the need to study the natural infection of Ny. neivai and other species in VL endemic areas, regardless of the presence of Lu. longipalpis.Thus, this study used the multiplex polymerase chain reaction (PCR) methodology and sequencing in sand flies captured in the municipality of Itaperuçu, located in Vale do Ribeira, Paraná, to evaluate the natural infection in sand flies.They were sorted by external morphology, sexed, and mounted for species-level identification, kept in 80% alcohol, and conserved at -20ºC for extraction and sequencing.The sand flies were identified using the proposal of Galati (12) and the abbreviation of the genera proposed by Marcondes. (13)A extraction and multiplex PCR -A total of 1,186 sand fly specimens were grouped into 176 pools of 2 to 10 specimens each per collection.DNA extraction was performed using the commercial Wizard SV Genomic DNA Purification System kit (PROMEGA TM , Madison, WI, USA), following the manufacturer's specifications. (14)The Multiplex PCR was designed to simultaneously amplify the cacophony gene in the IVS6 region in sand flies of the neotropical genus Lutzomyia (as an internal control for polymerase enzyme activity and DNA extraction) and the conserved minicircle region of DNA from the kinetoplast of Leishmania spp.For contamination control, all instruments and work areas were decontaminated with a diluted chloride solution and ultraviolet light.

Collection
Cloning and sequencing -DNA recovered from each Leishmania positive sample was subjected to a second semi-nested-PCR assay targeting the hsp70 gene.This validated the gene region to distinguish different species of Leishmania present in Brazil.In the first round of PCR, a 234 bp fragment of hsp70 was amplified using the oligonucleotides 5'-GGA CGA GAT CGA GCG CAT GGT-3' and 5'-TCC TTC GAC GCC TCC TGG TTG-3'.In the second round, the same forward oligonucleotide is paired with the following reverse oligonucleotide: 5'-GGA GAA CTA CGC GTA CTC GAT GAA G-3' to amplify an internal 144 bp region of the 234 bp fragment.The amplified fragments were purified and cloned into competent Escherichia coli DH5α cells using the vector from the pGEM T-Easy Vector kit (Promega TM ), according to the manufacturer's recommendations.
Sanger sequencing was performed using the RPT01A-PDTIS sequencing platform, Fiocruz-RJ (ABI 3730XL Applied Biosystem), with the Big Dye Terminator v3.1 Cycle Sequencing Ready Reaction kit (Applied Biosystems, CA, USA).The electropherograms were analysed using the Phred program, and the regions with good sequence resolution were submitted to assembly using the CAP3 program, removing the vector sequence using the NCBIVecScreen program (http:// www.ncbi.nlm.nih.gov/VecScreen/VecScreen.html).The sequences were compared with those available in the BLASTnucleotide database (http://blast.ncbi.nlm.nih.gov/Blast.cgi)using the BLASTN algorithm.

RESULTS AND DISCUSSION
Between May 2017 and July 2018, a total of 1,186 specimens were collected, and, from these, 176 pools of sand fly specimens were isolated to be used for the DNA research of Leishmania spp.Among the analysed species, Ny. neivai was the most abundant (88.62%), followed by Pintomyia fischeri (9.2%) and Mg.migonei (1.18%).Brumptomyia troglodytes and Expapillata firmatoi to-Municipality of Itaperuçu, Vale do Ribeira, Paraná, Brazil.
gether totalised 1.01% of the total number of females analysed.There was a remarkable increase in Ny. neivai in September and February, which were the warmest and least humid months of the experimental period.This species was observed in animal shelters and indoors, proving its adaptation to modified environments.
Three pools of Ny. neivai tested positive for Leishmania spp.DNA by PCR.Sequencing confirmed two as L. braziliensis and one as L. infantum.In the Vale do Ribeira, an endemic area for ATL, the detection of Ny. neivai specimens with L. braziliensis DNA further supports the presence of this parasite in the region.It indicates that this species may play a central role in transmitting the parasite.Several factors demonstrate the possible vectorial role of Ny. neivai for the etiologic agent of ATL in southern Brazil, such as the very low density of other suspected vectors for L. braziliensis (15,16) and the high population density of Ny. neivai.Other factors include the constant monthly frequency of this sand fly, combined with a marked seasonality and a clear adaptation to anthropic environments, which present environmental conditions favourable to the persistence of the enzootic cycle of L. braziliensis.
The discovery of L. infantum DNA introduces a novel aspect to this region.Previously, the circulation of this parasite had only been reported in the municipality of Foz do Iguaçu. (11)Vector species are geographically distributed in their respective transmission cycles, adapted to the abiotic factors of that habitat.However, as external interactions such as anthropic, climatic, and spatial actions start to act in that originally preserved environment, the distribution of these species can narrow the relationship between the etiological agent and other vectors that are not part of their natural cycle. (17)ven though the determination of vector competence can only be described after observing the protozoa inside the vector's gut, the presence of Leishmania spp. in sand flies that are not incriminated as vectors is extremely important. (14)he combination of these factors, along with the findings detailed in our study, serves as a warning regarding the spread of VL in the state.This is particularly significant as Ny.neivai has previously been linked to the transmission of L. infantum in the Southeast and Southern regions of Brazil.The first report of natural infection of the species by L. infantum was described by Saraiva et al. (18) in an area with no registered cases of VL, in the State of Minas Gerais.In the second report conducted by Dias et al. (4) in the municipality of Florianópolis, where 11 species of sand flies were collected, with three testing positive for Leishmania sp.However, the confirmation of L. infantum infection was exclusively observed in Ny. neivai.
The Ny. neivai's positivity for L. infantum DNA is described in the first report in the State of Paraná, in a municipality that does not have Lu.longipalpis in its sand fly fauna, nor cases of VL.In addition, other vectors suspected of transmitting L. infantum, Pi. fischeri (3) and Mg.migonei (2,19) were collected in our study, but without positive pools for Leishmania DNA.
Our findings indicate the circulation of L. infantum in an area traditionally considered free from VL and reinforce the importance of entomological and health surveillance in this region.
, packaging and assembly of sand flies -Collections were carried out using modified CDC light traps and Shannon traps (Shannon, 1939) over two years (from June/2016 to July/2018) in the municipality of Itaperuçu (Figure).In a period of 24 months, the CDC traps were installed in the peridomicile of 10 residences for 12 h a day, for three consecutive days.Shannon traps were placed in a forested area for 3 h once a month.For natural infection analysis, specimens from the period between May 2017 and July 2018 were considered.Ny. neivai specimens were captured by the respective traps both in peridomicile and forested areas.The traps were installed following the recommendations of the National Leishmaniasis Control Program of the Brazilian Ministry of Health.The captured sand flies were transported to the Laboratory of Molecular Parasitology of the Department of Basic Pathology, Sector of Biological Sciences at the Federal University of Paraná (DPAT/BL/UFPR).